In general, this invention relates to typing and matching human leukocyte antigens or alleles of human leukocyte antigens and in particular, to high throughput screening methods of human leukocyte antigen matching or alleles of human leukocyte antigens.
The human leukocyte antigen complex (also known as the major histocompatibility complex) spans approximately 3.5 million base pairs on the short arm of chromosome 6. It is divisible into 3 separate regions which contain the class I, the class II and the class III genes. In humans, the class I HLA complex is about 2000 kb long and contains about 20 genes. Within the class I region exist genes encoding the well characterized class I MHC molecules designated HLA-A, HLA-B and HLA-C. In addition, there are nonclassical class I genes that include HLA-E, HLA-F, HLA-G, HLA-H, HLA-J and HLA-X as well as a new family known as MIC. The class II region contains three genes known as the HLA-DP, HLA-DQ and HLA-DR loci. These genes encode the xcex1 and xcex2 chains of the classical class II MHC molecules designated HLA-DR, DP and DQ. In humans, nonclassical genes designated DM, DN and DO have also been identified within class II. The class III region contains a heterogeneous collection of more than 36 genes. Several complete components are encoded by three genes including TNF-xcex1 and TNF-xcex2.
Any given copy of chromosome 6 can contain many different alternative versions of each of the preceding genes and thus can yield proteins with distinctly different sequences. The loci constituting the MHC are highly polymorphic, that is, many forms of the gene or alleles exist at each locus. Several hundred different allelic variants of class I and class II MHC molecules have been identified in humans. However, any one individual only expresses up to 6 different class I molecules and up to 12 different class II molecules.
The foregoing regions play a major role in determining whether transplanted tissue will be accepted as self (histocompatible) or rejected as foreign (histoincompatible). For instance, within the class II region, three loci i.e., HLA-DR, DQ and DP are known to express functional products. Pairs of A and B genes within these three loci encode heterodimeric protein products which are multi-allelic and alloreactive. In addition, combinations of epitopes on DR and/or DQ molecules are recognized by alloreactive T cells. This reactivity has been used to define xe2x80x9cDwxe2x80x9d types by cellular assays based upon the mixed lymphocyte reaction (MLR). It has been demonstrated that matching of donor and recipient HLA-DR and DQ alleles prior to allogeneic transplantation has an important influence on allograft survival. Therefore, HLA-DR and DQ matching is now generally undertaken as a clinical prerequisite for renal and bone marrow transplantation as well as cord blood applications.
Until recently, matching has been confined to serological and cellular typing. For instance, in the microcytotoxicity test, white blood cells from the potential donor and recipient are distributed in a microtiter plate and monoclonal antibodies specific for class I and class II MHC alleles are added to different wells. Thereafter, complement is added to the wells and cytotoxicity is assessed by uptake or exclusion to various dyes by the cells. If the white blood cells express the MHC allele for a particular monoclonal antibody, then the cells will be lysed on addition of complement and these dead cells will take up the dye. (see, Terasaki and McClelland, (1964) Nature, 204:998). However, serological typing is frequently problematic, due to the availability and crossreactivity of alloantisera and because live cells are required. A high degree of error and variability is also inherent in serological typing, which ultimately affects transplant outcome and survival (Sasazuki et al., (1998) New England J. of Medicine 339: 1177-1185). Therefore, DNA typing is becoming more widely used as an adjunct, or alternative, to serological tests.
Initially, the most extensively employed DNA typing method for the identification of these alleles has been restriction fragment length polymorphism (RFLP) analysis. This well established method for HLA class II DNA typing suffers from a number of inherent drawbacks. RFLP typing is too time-consuming for clinical use prior to cadaveric renal transplantation for example, and for this reason it is best suited to live donor transplantation or retrospective studies. Furthermore, RFLP does not generally detect polymorphism within the exons which encode functionally significant HLA class II epitopes, but relies upon the strong linkage between alleles-specific nucleotide sequences within these exons and restriction endonuclease recognition site distribution within surrounding, generally noncoding, DNA.
In addition to restriction fragment length polymorphism (PCR-RFLP), an even more popular approach has been the hybridization of PCR amplified products with sequence-specific oligonucleotide probes (PCR-SSO) to distinguish between HLA alleles (see, Tiercy et al., (1990) Blood Review 4: 9-15). This method requires a PCR product of the HLA locus of interest be produced and then dotted onto nitrocellulose membranes or strips. Then each membrane is hybridized with a sequence specific probe, washed, and then analyzed by exposure to x-ray film or by colorimetric assay depending on the method of detection. Similar to the PCR-SSP methodology, probes are made to the allelic polymorphic area responsible for the different HLA alleles. Each sample must be hybridized and probed at least 100-200 different times for a complete Class I and II typing. Hybridization and detection methods for PCR-SSO typing include the use of non-radioactive labeled probes, microplate formats, etc. (see e.g., Saiki et al. (1989) Proc. Natl. Acad. Sci., U.S.A. 86: 6230-6234; Erlich et al. (1991) Eur. J. Immunogenet. 18(1-2): 33-55; Kawasaki et al. (1993) Methods Enzymol. 218:369-381), and automated large scale HLA class II typing. A common drawback to these methods, however, is the relatively long assay times neededxe2x80x94generally one to two daysxe2x80x94and their relatively high complexity and resulting high cost. In addition, the necessity for sample transfers and washing steps increases the chances that small amounts of amplified DNA might be carried over between samples, creating the risk of false positives.
More recently, a molecular typing method using sequence specific primer amplification (PCR-SSP) has been described (see, Olerup and Zetterquist (1992) Tissue Antigens 39: 225-235). This PCR-SSP method is simple, useful and fast relative to PCR-SSO, since the detection step is much simpler. In PCR-SSP, allelic sequence specific primers amplify only the complementary template allele, allowing genetic variability to be detected with a high degree of resolution. This method allows determination of HLA type simply by whether or not amplification products (collectively called an xe2x80x9campliconxe2x80x9d) are present or absent following PCR. In PCR-SSP, detection of the amplification products is usually done by agarose gel electrophoresis followed by ethidium bromide (EtBr) staining of the gel. Unfortunately, the electrophoresis process takes a long time and is not very suitable for large number of samples, which is a problem since each clinical sample requires testing for many potential alleles. Gel electrophoresis also is not easily adapted for automatic HLA-DNA typing.
Another HLA typing method is SSCPxe2x80x94Single-Stranded Conformational Polymorphism. Briefly, single stranded PCR products of the different HLA loci are run on non-denaturing Polyacrylamide Gel Electrophoresis (PAGE). The single strands will migrate to a unique location based on their base pair composition. By comparison with known standards, a typing can be deduced. It is the only method that can determine true homozygosity. However, many PAGE have to be run and many controls have to be run to make it a viable typing method. This method is very time consuming, labor intensive, and not really suited for large volume analysis.
In view of the foregoing, what is needed in the art is a method of determining genomic information from a highly polymorphic system such as the HLA class I and class II regions. The present invention provides a highly accurate and efficient HLA class I and class II sequence-based typing method that is rapid, reliable and completely automatable.
The present invention provides new and improved methods for HLA typing. In addition, the methods eliminate the reliance on agarose gel electrophoresis usage for the sequence specific primer (SSP) method for performing HLA DNA typing and obviates the reliance on using cumbersome blot membranes for sequence-specific oligonucleotide probe hybridization (SSO) as well as many of the human errors associated with manual interpretation of bands and assignment of alleles. Thus, the methods of the present invention decrease significantly the number of human errors and the amount of time and effort it takes to perform DNA HLA typing.
In certain aspects, the present invention provides a method of detecting amplified DNA in which the risks of sample cross-contamination and resulting false positive results are reduced. In addition, the present invention provides methods that can allow for reliable, rapid analysis of multiple samples. Moreover, the present invention provides a method of detecting amplified DNA that is relatively simple, and results in a relatively low cost per analysis and is amenable to automation and high throughput matching.
In one aspect, the present invention provides methods for identifying an HLA genotype of a subject. The method involves (a) obtaining a sample containing a template nucleic acid from said subject; (b) amplifying the template nucleic acid with a plurality of HLA allele-specific forward primers and HLA allele-specific reverse primers to form amplification products, wherein the forward primers or reverse primers comprise a detectable label; (c) hybridizing the amplification products with a plurality of HLA locus-specific capture oligonucleotides immobilized on a solid phase to form a plurality of detectable complexes; and (d) detecting the detectable complexes to identify the HLA genotype of the subject.
Another aspect of the present invention provides methods for identifying an HLA genotype of a subject that involves (a) obtaining a sample containing a template nucleic acid from the subject; (b) amplifying the template nucleic acid with a plurality of HLA allele-specific forward primers and HLA allele-specific reverse primers to form amplification products, wherein the forward primers or reverse primers contain a detectable label; (c) hybridizing the amplification products with a plurality of HLA locus-specific capture oligonucleotides to form a plurality of detectable complexes; (d) immobilizing the detectable complexes on a solid phase; and (e) detecting the detectable complexes to identify the HLA genotype of the subject.
In yet another aspect of the invention, methods for identifying an HLA genotype of a subject is provided that involves: immobilizing a plurality of HLA allele-specific reverse primers on a solid phase; amplifying the template nucleic acid with a plurality of HLA allele-specific forward primers and the immobilized reverse HLA allele-specific reverse primers to form amplification products; and detecting the amplification products to identify the HLA genotype of the subject.
In certain embodiments of the present invention, template nucleic acid that is isolated from blood or cord blood is amplified. The template nucleic acid can be any gene derived sequences, including, but not limited to cDNA and genomic DNA.
In certain embodiments, oligonucleotides are immobilized on a solid phase. Examples of solid phase include, but are not limited to: a bead, a chip, a microtiter plate, a polycarbonate microtiter plate, polystyrene microtiter plate, and a slide. The methods of the present invention can be also used to determine class I and class II HLA genotypes. In certain embodiments, HLA allele-specific forward primers and HLA allele-specific reverse primers are used to amplify the template nucleic acid to generate amplification products. In some embodiments, the HLA allele-specific primers are selected from primers denoted as SEQ ID NOS:1-160 and SEQ ID NOS: 169-269.
In some embodiments of the invention, capture oligonucleotides are employed. In certain preferred embodiments, locus-specific capture oligonucleotides are used in the HLA genotyping methods and can be selected from the primers such as SEQ ID NOS: 272-277 and SEQ ID NOS:165-168. The capture oligonucleotides can be modified with a moiety that aids in immobilizing the capture oligonucleotide to a solid phase. In certain embodiments, moieties such as a 5xe2x80x2 amine group or a 5xe2x80x2(T)5-20 oligonucleotide sequence are utilized.
Detectable labels can be used with certain embodiments of the present invention. Examples of a detectable label, include, but are not limited to a radioactive moiety, a fluorescent moiety, a chemiluminescent moiety, an antigen, or a binding protein. In certain embodiments, fluorescent moieties such as fluorescein or 5-(2xe2x80x2-aminoethyl)aminonaphtalene-1-sulfonic acid (EDANS) are attached to oligonucleotides to facilitate detection.
These embodiments as well as additional objects and advantages will become more readily apparent when read with the accompanying FIGURE and detailed description which follows.
An xe2x80x9callelexe2x80x9d is one of the different nucleic acid sequences of a gene at a particular locus on a chromosome. One or more genetic differences can constitute an allele. Examples of HLA allele sequences are set out in Mason and Parham (1998) Tissue Antigens 51: 417-66, which list HLA-A, HLA-B, and HLA-C alleles and Marsh et al. (1992) Hum. Immunol. 35:1, which list HLA Class II alleles for DRA, DRB, DQA1, DQB1, DPA1, and DPB1.
A xe2x80x9clocusxe2x80x9d is a discrete location on a chromosome that constitutes a gene. Exemplary loci are the class I MHC genes designated HLA-A, HLA-B and HLA-C; nonclassical class I genes including HLA-E, HLA-F, HLA-G, HLA-H, HLA-J and HLA-X, MIC; and class II genes such as HLA-DP, HLA-DQ and HLA-DR.
A method of xe2x80x9cidentifying an HLA genotypexe2x80x9d is a method that permits the determination or assignment of one or more genetically distinct HLA genetic polymorphisms.
The term xe2x80x9camplifyingxe2x80x9d refers to a reaction wherein the template nucleic acid, or portions thereof, are duplicated at least once. Unless specifically stated xe2x80x9camplifyingxe2x80x9d may refer to arithmetic, logarithmic, or exponential amplification. The amplification of a nucleic acid can take place using any nucleic acid amplification system, both isothermal and thermal gradient based, including but not limited to, polymerase chain reaction (PCR), reverse-transcription-polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), self-sustained sequence reaction (3SR), and transcription mediated amplifications (TMA). Typical nucleic acid amplification mixtures (e.g., PCR reaction mixture) include a nucleic acid template that is to be amplified, a nucleic acid polymerase, nucleic acid primer sequence(s), and nucleotide triphosphates, and a buffer containing all of the ion species required for the amplification reaction.
An xe2x80x9camplification productxe2x80x9d is a single stranded or double stranded DNA or RNA or any other nucleic acid products of isothermal and thermal gradient amplification reactions that include PCR, TMA, 3SR, LCR, etc.
The phrase xe2x80x9ctemplate nucleic acidxe2x80x9d refers to a nucleic acid polymer that is sought to be copied or amplified. The xe2x80x9ctemplate nucleic acid(s)xe2x80x9d can be isolated or purified from a cell, tissue, animal, etc. Alternatively, the xe2x80x9ctemplate nucleic acid(s)xe2x80x9d can be contained in a lysate of a cell, tissue, animal, etc. The template nucleic acid can contain genomic DNA, cDNA, plasmid DNA, etc.
An xe2x80x9cHLA allele-specificxe2x80x9d primer is an oligonucleotide that hybridizes to nucleic acid sequence variations that define or partially define that particular HLA allele.
An xe2x80x9cHLA locus-specificxe2x80x9d primer is an oligonucleotide that permits the amplification of a HLA locus sequence or that can hybridize specifically to an HLA locus.
A xe2x80x9cforward primerxe2x80x9d and a xe2x80x9creverse primerxe2x80x9d constitute a pair of primers that can bind to a template nucleic acid and under proper amplification conditions produce an amplification product. If the forward primer is binding to the sense strand then the reverse primer is binding to antisense strand. Alternatively, if the forward primer is binding to the antisense strand then the reverse primer is binding to sense strand. In essence, the forward or reverse primer can bind to either strand as long as the other reverse or forward primer binds to the opposite strand.
The term xe2x80x9cdetectable labelxe2x80x9d refers to a moiety that is attached through covalent or non-covalent means to an oligonucleotide. A xe2x80x9cdetectable labelxe2x80x9d can be a radioactive moiety, a fluorescent moiety, a chemiluminescent moiety, etc.
The term xe2x80x9cfluorescent labelxe2x80x9d refers to label that accepts radiant energy of one wavelength and emits radiant energy of a second wavelength.
The phrase xe2x80x9chybridizingxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence or subsequence through specific binding of two nucleic acids through complementary base pairing. Hybridization typically involves the formation of hydrogen bonds between nucleotides in one nucleic acid and complementary sequences in the second nucleic acid.
The phrase xe2x80x9chybridizing specificallyxe2x80x9d refers to hybridizing that is carried out under stringent conditions.
The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a capture oligonucleotide, oligonucleotide or amplification product will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the capture oligonucleotides are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is at most about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. An extensive guide to the hybridization and washing of nucleic acids is found in Tijssen (1993) Laboratory Techniques in biochemistry and molecular biologyxe2x80x94hybridization with nucleic acid probes parts I and II, Elsevier, N.Y., and, Choo (ed) (1994) Methods In Molecular Biology Volume 33xe2x80x94In Situ Hybridization Protocols Humana Press Inc., New Jersey; Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Current Protocols in Molecular Biology (Ausubel et al., eds., (1994)).
The term xe2x80x9ccomplementary base pairxe2x80x9d refers to a pair of bases (nucleotides) each in a separate nucleic acid in which each base of the pair is hydrogen bonded to the other. A xe2x80x9cclassicalxe2x80x9d (Watson-Crick) base pair always contains one purine and one pyrimidine; adenine pairs specifically with thymine (A-T), guanine with cytosine (G-C), uracil with adenine (U-A). The two bases in a classical base pair are said to be complementary to each other.
xe2x80x9cBind(s) substantiallyxe2x80x9d refers to complementary hybridization between a capture nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
The term xe2x80x9ccapture oligonucleotidexe2x80x9d refers to a nucleic acid sequence or nucleic acid subsequence that can hybridize to another oligonucleotide, amplification product, etc. and has the ability to be immobilized to a solid phase. A capture oligonucleotide typically hybridizes to at least a portion of an amplification product containing complementary sequences under stringent conditions.
A xe2x80x9cHLA locus-specific capture oligonucleotidexe2x80x9d is a capture oligonucleotide that is complementary to and hybridizes to a conserved region of an HLA locus. For example a xe2x80x9cHLA locus-specific capture oligonucleotidexe2x80x9d that is specific for the HLA-A locus will hybridize to one or more conserved regions or subsequences of the HLA-A locus.
A compound is xe2x80x9cimmobilized on a solid phasexe2x80x9d when it is directly or indirectly attached to the solid phase. Such immobilization may be through covalent and/or non-covalent bonds.
The term xe2x80x9ccorresponding nucleotide,xe2x80x9d is used to refer to the position of a nucleotide in a first nucleic acid by reference to a second nucleic acid. Thus, a corresponding nucleotide refers to a nucleotide that it is positionally located opposite to a base where neighboring bases are all hybridized pairs.
xe2x80x9cSubsequencexe2x80x9d refers to a sequence of nucleic acids that comprise a part of a longer sequence of nucleic acids.
The term xe2x80x9cportionsxe2x80x9d should similarly be viewed broadly, and would include the case where a xe2x80x9cportionxe2x80x9d of a DNA strand is in fact the entire strand.
The term xe2x80x9cspecificityxe2x80x9d refers to the proportion of negative test results that are true negative test result. Negative test results include false positives and true negative test results.
The term xe2x80x9csensitivityxe2x80x9d is meant to refer to the ability of an analytical method to detect small amounts of analyte. Thus, as used here, a more sensitive method for the detection of amplified DNA, for example, would be better able to detect small amounts of such DNA than would a less sensitive method. xe2x80x9cSensitivityxe2x80x9d refers to the proportion of expected results that have a positive test result.
The term xe2x80x9creproducibilityxe2x80x9d as used herein refers to the general ability of an analytical procedure to give the same result when carried out repeatedly on aliquots of the same sample.
The term xe2x80x9campliconxe2x80x9d is used herein to mean a population of DNA molecules that has been produced by amplification, e.g., by PCR.
The term xe2x80x9cmolecular beacon,xe2x80x9d as used herein refers to a molecule capable of participating in a specific binding reaction and whose fluorescence activity changes when the molecule participates in that binding reaction.
I. Introduction
The present invention provides methods for HLA genotyping of human leukocyte antigens, as well as other molecular diagnostic protocols relating to the detection of DNA sequences and sequence variations using nucleic acid amplification methods. Advantageously, the methods described herein can be used to detect genetic mutations, detect cancer gene mutations, microbial and cancer drug resistance mutations, detection of viruses, bacteria, fungi, parasites and any other microbes, forensics, parentage, etc.
In particular, the methods of the present invention are useful for determining HLA genotypes of samples from subjects. Such genotyping is important in the clinical arena for the diagnosis of disease, transplantation of organs, and bone marrow and cord blood applications.
In the present invention, allelic-specific HLA primers are used to amplify HLA sequences. In some embodiments, these amplification products can be immobilized to a solid phase using a locus-specific or an allele-specific capture oligonucleotide. In certain embodiments, the locus-specific capture oligonucleotides are preferred as fewer capture oligonucleotides need to be generated to carry out the HLA genotyping. In other embodiments, one HLA-specific primer is immobilized to a solid phase and the target is amplified using another HLA-specific primer that is free in solution. The advantages and details for carrying out the present invention will be discussed more fully below.
II. Materials Used in the Present Invention
Oligonucleotides
Oligonucleotides used in the present invention (e.g., allele and locus-specific oligonucleotides) can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, (1981) Tetrahedron Letts. 22:1859-1862, using an automated synthesizer, as described in Van Devanter et al., (1984) Nucleic Acids Res. 12: 6159-6168. Purification of oligonucleotides is typically by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Reanier, (1983) J. Chrom. 255:137-149.
HLA allele-specific Primers
The HLA allele-specific primers used in the present invention are designed to amplify HLA allele sequences. Since 1995, 213 class I (HLA-A, HLA-B, and HLA-C) and 256 class II (HLA-DR, HLA-DP, and HLA-DQ) alleles had been identified and sequenced (see e.g., Krausa and Browning (1996) Detection of HLA gene polymorphism in Browning M, McMichael A, ed. HLA and MHC: genes, molecules and function. Oxford: Bios Scientific Publishers Limited, pp. 113-138), with new alleles being discovered all the time. The sequences of many of these alleles are publicly available through GenBank and other gene databases and have been published (see e.g., Mason and Parham (1998) Tissue Antigens 51: 417-66, listing HLA-A, HLA-B, and HLA-C alleles; Marsh et al. (1992) Hum. Immunol. 35:1, listing HLA Class II allelesxe2x80x94DRA, DRB, DQA1, DQB1, DPA1, and DPB1). Also, the use of allele-specific primers (sequence-specific primers (SSP)) has permitted the specific amplification of HLA allele sequences (see e.g., Bunce and Welsh (1994) Tissue Antigens 43: 7-17, amplification of HLA-C alleles; Bunce et al. (1995) Tissue Antigens 46: 355-67, amplification of HLA-A.B.C. DRB1, DRB3, DRB4, DRB5 and DQB1 alleles with sequence-specific primers; Gilchrist et al. (1998) Tissue Antigens 51: 51-61, HLA-DP typing with sequence specific primers).
In the design of the HLA primer pairs for the primer mixes, primers were selected based on the published HLA sequences available in the literature. A chart of the HLA alleles and sequences was examined and the polymorphic sites were identified. Then pairs of primers were selected that would produce PCR products to a group of HLA alleles. The sequence specific nucleic acid amplification reaction typically uses at least a pair of PCR primers for each allele, both of which contain the discriminating sequences with at least one or more of the changed nucleotides at the 3xe2x80x2 end of each PCR primer. Since the 3xe2x80x2 end is the end where polymerization takes place, if a mismatch occurs due to sequence non-complementarily, nucleic acid amplification will take place and one would not expect a xe2x80x9cfalse positive.xe2x80x9d However, if a match occurs, then the amplification can proceed. For example, HLA class I allele-specific primers and HLA class II allele-specific primers are listed in Table 1 (SEQ ID NOS: 1-160) and 2 (SEQ ID NOS: 169-269), respectively. Examples of control primers listed in Table 1 are CI53 (SEQ ID NO: 161), CI54 (SEQ ID NO: 162), CI148 (SEQ ID NO: 163), and CI149 (SEQ ID NO: 164). Examples of control primers listed in Table 2 are DPA-E(PC) (SEQ ID NO: 270), and DPA-F (PC) (SEQ ID NO: 271). The Class I primers are selected to amplify Class I exon 2 and exon 3 products. The Class II primers are selected to amplify Class II exon 2 products. In certain embodiments, the primers listed in Tables 3 and 4 are used as exemplary groups of primer pairs and the HLA specificities these pairs can identify after successful positive PCR amplifications with the appropriate DNA templates for HLA class I and II alleles respectively. By utilizing a pair of primers, each PCR reaction identifies two sites of polymorphism and therefore increases the specificity of the reaction. Those of skill in the art will recognize a multitude of oligonucleotide compositions that can be used as HLA allele-specific primers to specifically amplify HLA allele sequences. In addition, customized sets of HLA-specific primers can be created to cater to detection of the allele distribution for various ethnicities or racial groups by simply changing the primer pair combinations. In this manner, detection of new alleles can be easily added to the methods of the present invention.
Capture Oligonucleotides
In certain embodiments, the invention involves locus-specific capture oligonucleotides or allele-specific capture oligonucleotides. Locus-specific oligonucleotide can hybridize to a conserved region in a HLA locus; a locus-specific capture oligonucleotide has the ability to hybridize to some or all of the sequences that can be generated by the amplification of HLA allele sequences using HLA-specific primers. Locus-specific sequences have been identified in HLA loci. For example, locus-specific sequences for HLA-class I genes have been delineated in the first and third introns flanking the polymorphic second and third exons (see e.g., Cereb et al. (1995) Tissue Antigens 45: 1-11). The capture oligonucleotides should be of such length and composition so as to be able to hybridize with the allele-specific PCR products. In certain embodiments, HLA locus-specific class I capture oligonucleotides contain the following sequences: for HLA-A (CICptA1, Class I Capture Oligo A1, 5xe2x80x2ACGCCTACGACGGCAAGGATTACATCGCCC3xe2x80x2 (SEQ ID NO:165); and CICptA2, Class I Capture Oligo A2, 5xe2x80x2GATGGAGCCGCGGTGGATAGAGCAGGAGGG3xe2x80x2(SEQ ID NO:166), for HLA-B (CICptB1, Class I Capture Oligo B1, 5xe2x80x2CAGTTCGTGAGGTTCGACAGCGACGCC3xe2x80x2(SEQ ID NO:167), and CICptB2, Class I Capture Oligo B2, 5xe2x80x2CTGCGCGGCTACTACAACCAGAGCGAGGCC3xe2x80x2(SEQ ID NO:168). In other embodiments, HLA locus-specific class II capture oligonucleotides contain the following sequences: for HLA-DQ (DQCPT1, 5xe2x80x2CACGTCGCTGTCGAAGCGCACGTACTCCTC3xe2x80x2 (SEQ ID NO:272); DQCPT2, 5xe2x80x2CACGTCGCTGTCGAAGCGGACGATCTCCTT3xe2x80x2 (SEQ ID NO:273); DQCPT3, 5xe2x80x2CACGTCGCTGTCGAAGCGTGCGTACTCCTC3xe2x80x2 (SEQ ID NO:274); DQCPT4, 5xe2x80x2CACGTCGCTGTCGAAGCGCGCGTACTCCTC3xe2x80x2 (SEQ ID NO:275); and DQCPT5, 5xe2x80x2CACGTCGCTGTCGAAGCGCACGTCCTCCTC3xe2x80x2(SEQ ID NO:276), for HLA-DR (DRCPT1, DRCP, 5xe2x80x2TGGCGTGGGCGAGGCAGGGTAACTTCTTTA3xe2x80x2 (SEQ ID NO:277)). In certain embodiments, it may require the use of more than one capture oligonucleotide to hybridize to all of the HLA allele amplification products.
Modification of Oligonucleotides
In certain embodiments of the present invention, oligonucleotides are modified or synthesized as modified oligonucleotides to facilitate immobilization or detection.
In certain embodiments, where capture oligonucleotides are used or where an immobilized amplification primer is used, it is desirable to modify the particular oligonucleotide to affix it to a solid phase or support. It is desired that the modification of the capture oligonucleotide does not interfere with its ability to bind to an HLA allele-specific amplification product. Those of skill in the art will recognize a variety of methods to immobilize oligonucleotides to a solid phase. For example, oligonucleotides can be directly or indirectly immobilized on a solid phase. The oligonucleotides can be immobilized directly to the solid phase through covalent and non-covalent bonds. For example, the 5xe2x80x2 end of an oligonucleotide can be synthesized with an amine moiety (see Kawasaki et al. (1993)). In certain embodiments, an amine moiety with a C6 carbon spacer is conjugated to the 5xe2x80x2 end of a capture oligo or amplification primer. The amine-modified primers are affixed to the surface of a substrate such a Biodyne C membrane (Pall Biosupport) (Kawasaki et al. (1993)) or through a commercially available microtiter plate (e.g., Xenobind(trademark) (Covalent Binding Microwell Plates), Xenopore, Hawthorne, N.J.). Alternatively a polythymidine (polyT) stretch can be added to an oligonucleotide by terminal deoxyribonucletotidyltransferase (Saiki et al. (1989)). Such a polyT stretch can be fixed to many solid substrates (e.g., nylon) using UV light leaving the rest of the oligonucleotide free to hybridize to another nucleic acid. Preferably, the polyT stretch is from 5 to 20 T""s.
Alternatively, the oligonucleotides can be indirectly bound to the solid phase by coating the solid phase with a substance or molecule that can bind to the oligonucleotides. Biotinylated oligonucleotides can also be used as capture oligonucleotides. Methods are known in the art for synthesizing biotinylated oligonucleotide (e.g., by synthesizing a primer with a biotinylated 5xe2x80x2 end nucleotide as the terminal residue) (see e.g., Innis et al. (1990)). Biotinylated oligonucleotides can be affixed to a substrate that is coated with avidin.
A high density array of capture oligonucleotides or amplification primers can be also synthesized on a substrate by attaching photoremovable groups to the surface of a substrate, exposing selected regions of the substrate to light to activate those regions, attaching a nucleic acid monomer with a photoremovable group to the activated regions, and repeating the steps of activation and attachment until probes of the desired length and sequences are synthesized. (See, e.g., Fodor et al. (1991) Science 251: 767-773 and U.S. Pat. No. 5,143,854). The resulting array of oligonucleotides can then be used to in the methods of the present invention.
A variety of solid supports or phases can be used in the present invention. Examples of solid supports include, without limitation, bead, microtiter plates, and chips. Beads can be composed of materials such as Sepharose, agarose, polystyrene, etc. and can be paramagnetic. Microtiter plates are commercially available in a variety of formats (e.g., 96, 384 and 1536 well plates) and materials (e.g., polystyrene). The plates can be either polycarbonate plates in which case the thermal gradient nucleic acid amplification reaction (such as PCR) can happen directly in the well or polystyrene in which case the thermal gradient nucleic acid amplification reaction (such as PCR) has to take place in a separate polycarbonate plate and transferred to the surface modified and oligonucleotide attached plate. Isothermal nucleic acid amplification methods can be conducted in polystyrene plates. chips can be comprised of a variety of materials, layers and substrates. Polymers which may be used as solid supports or phases include, but are not limited to, the following: polystyrene; poly(tetra)fluoroethylene (PTFE); polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate; polyvinylethylene; polyethyleneimine; poly(etherether)ketone; polyoxymethylene (POM); polyvinylphenol; polylactides; polymethacrylimide (PMI); polyatkenesulfone (PAS); polypropylene; polyethylene; polyhydroxyethylmethacrylate (HEMA); polydimethylsiloxane; polyacrylamide; polyimide; and block-copolymers. The solid support on which an oligonucleotide resides may also be a combination of any of the aforementioned solid support materials.
Oligonucleotides Containing Detectable Labels
Detectable labels can also be attached to oligonucleotides to facilitate detection of the oligonucleotide in an analyte. Detectable labels can be detected either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radiolabels (e.g., 3H, 13C, 14C, 32P, 35S, 125I, etc.), fluorescent dyes, fluorophores, fluorescent moieties, chemiluminescent moieties, electron-dense reagents, enzymes and their substrates (e.g., as commonly used in enzyme-linked immunoassays, e.g., alkaline phosphatase and horse radish peroxidase), biotin-streptavidin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. The label or detectable moiety is typically bound, either covalently, through a linker or chemical bound, or through ionic, van der Waals or hydrogen bonds to the molecule to be detected.
The detectable label should be stable to the amplifications conditions used and should permit direct or indirect detection. Indirect detection often involves the presence of one or more detection reagents. For example, one detectable label, biotin can be detected using an avidin conjugate such as avidin conjugated to an enzyme such as peroxidase (e.g., HRP), and a colorimetric substrate for peroxidase (e.g., TMB). The formation of colorimetric product can easily be detected using a spectrophotometer. For example, in certain embodiments, the primers listed in Tables 5 and 6 are biotinylated.
In certain embodiments, oligonucleotides comprising a quencher and a fluorophore moiety (molecular beacons) are contemplated. Molecular Beacons are single stranded oligonucleotide probes designed to have hairpin configuration by virtue of the presence of five to seven complementary nucleotides at their termini. The loop portion (10-40 nucleotides) is chosen so that the probe-amplification product hybrid is stable at the annealing temperature. The length of the arm sequences (5-7 nucleotides) is chosen so that a stem is formed at the annealing temperature of the polymerase chain reaction. Also the stem or arm sequence must be designed to ensure that the two arms hybridize to each other but not to the probe sequence. One end would carry a fluorophore (e.g. 5-(2xe2x80x2-aminoethyl)aminonaphtalene-1-sulfonic acid (EDANS) and the other a quencher (e.g. 4-(4xe2x80x2-dimethylaminophenylazo)benzoic acid (DABCYL). When a probe is not hybridized to its complementary target sequence, the hairpin folding reaction would take place and fluorescence does not occur due to quenching. Quenching occurs because the energy given off as light during fluorescence is transferred to the quencher and dissipated as heat. Since the energy is released as heat instead of light, the fluorescence is said to be quenched. However, if a complementary target sequence is present, hybridization to the target sequence would be favored over the internal hairpin due to the increased stability as a result of the longer stretches of complementary sequence. The hairpin would open up thus allowing for release of quenching and the probes to fluoresce. In the fluorophore-quencher pair example given above, when stimulated by UV light with a peak wavelength of 336 nm, EDANS emits a brilliant blue fluorescence with a peak wavelength of 490 nm. (Tyagi et al., (1996) Nature Biotechnology 14: 303-308; Tyagi et al. (1998) Nature Biotechnology 16:49-53; Paitek et al. (1998) Nature Biotechnology 16: 359-63; Kostrikis et al. (1998) Science 279:1228-1229).
III. Source of HLA Gene Sequences
The template HLA DNA sequences are contained in samples containing nucleic acid (e.g., DNA, RNA, etc.), which are obtained from a biological source. In certain embodiments, the nucleic acid is isolated from a biological source containing HLA gene sequences. The nucleic acid may be from any species having HLA gene sequences, which include but are not limited to, a human, a chimpanzee, a simian, a mouse, etc. Methods are known for lysing biological samples and preparing extracts or purifying DNA, RNA, etc. See, Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). In some embodiments, the biological source is blood, and is more preferably cord blood (e.g., blood from an umbilical cord). In methods involving cord blood or blood, two isolation procedures are preferred: Salt extraction with ethanol precipitation; and the Qiagen QIAamp(copyright) isolation method. For the salt extraction method, the cells are first lysed and centrifuged. Then water is added and the sample is centrifuged again. The pellet is digested with Proteinase K. The DNA is then extracted by the addition of 6M Guanidine HCl and incubation at 70xc2x0 C. for several minutes. The sample is centrifuged again and the supernatant is precipitated with cold 95% Ethanol. The pellet is then dried and resuspended in the appropriate buffer.
RNA template sequences that are amplified using the methods and compositions of the present invention may be a single RNA template or different RNA templates. The RNA can be isolated as total RNA from a cell, bacterium, virus etc. See, Ausubel et al. The total RNA may be subsequently purified as poly A+RNA or purified in a different manner to isolate certain species of interest. See Ausubel et al. Alternatively, the template RNA can be transcribed in vitro and used in the present invention. The RNA template sequence could also be reverse transcribed into cDNA and used as a nucleic acid template in the methods of the present invention.
IV. Amplification of HLA Gene Sequences from Nucleic Acid
The methods of the present invention involve the direct or indirect detection of HLA gene sequences that have been amplified from DNA or reverse transcribed DNA. To amplify the desired nucleic acid for HLA gene sequences, the following are usually present in the reaction vessel: template nucleic acid, nucleic acid polymerase, a molar excess of dNTPs, an antisense primer(s), and a sense-primer(s), for copying a HLA gene sequence from a template nucleic acid. Preferably, the reaction can be carried out in a thermal cycler oven to facilitate incubation times at the desired temperatures.
Reaction Components
The oligonucleotides that are used in the present invention, as well as oligonucleotides designed to detect amplification products, can be chemically synthesized as described above. These oligonucleotides can be labeled with radioisotopes, chemiluminescent moieties, or fluorescent moieties, etc. in a covalent or non-covalent manner. Such labels are useful for the characterization and detection of amplification products using the methods and compositions of the present invention.
Buffers that may be employed are borate, phosphate, carbonate, barbital, Tris, etc. based buffers. See Rose et al., U.S. Pat. No. 5,508,178. The pH of the reaction should be maintained in the range of about 4.5 to about 9.5. See U.S. Pat. No. 5,508,178. The standard buffer used in amplification reactions is a Tris based buffer between 10 and 50 mM with a pH of around 8.3 to 8.8. See Innis et al. (1990). In certain embodiments of the invention, a preferred buffer for the present invention is 150 mM Tris-HCl pH 8.8 for the amplification of class I HLA sequences and 20 mM Tris HCl pH 8.8 for class II HLA sequences.
The concentration of salt present in the reaction can affect the ability of primers to anneal to the template nucleic acid. See Innis et al. (1990). For example, potassium chloride can be added up to a concentration of about 50 mM to the reaction mixture to promote primer annealing. Sodium chloride can also be added to promote primer annealing. See Innis et al. (1990). In certain embodiments of the invention, the preferred salts are 30 mM Ammonium Chloride for class I HLA sequences and 100 mM KCl for class II sequences.
The concentration of magnesium ion in the reaction can be critical to amplifying the desired sequence(s). See Innis et al. (1990). Primer annealing, strand denaturation, amplification specificity, primer-dimer formation, and enzyme activity are all examples of parameters that are affected by magnesium concentration. See Innis et al. (1990). Amplification reactions should contain about a 0.5 to about a 5 mM magnesium concentration excess over the concentration of dNTPs. The presence of magnesium chelators in the reaction can affect the optimal magnesium concentration. A series of amplification reactions can be carried out over a range of magnesium concentrations to determine the optimal magnesium concentration. The optimal magnesium concentration can vary depending on the nature of the template nucleic acid(s) and the primers being used, among other parameters. In certain embodiments of the invention, the preferred magnesium concentrations are 4 mM MgCl2 and 3.4 mM MgCl2, for class I HLA sequences and class II HLA sequences, respectively.
Deoxyribonucleotide triphosphates (dNTPs) are added to the reaction to a final concentration of about 20 xcexcM to about 300 xcexcM. Each of the four dNTPs (G, A, C, T) should be present at equivalent concentrations. See Innis et al. In certain embodiments, 166 xcexcM dNTP is the preferred concentration of nucleotides.
A variety of DNA dependent polymerases are commercially available that will function using the methods and compositions of the present invention. For example, Taq DNA Polymerase may be used to amplify template DNA sequences. Also, a reverse transcriptase can be used in certain embodiments of the present invention. Reverse transcriptases, such as the thermostable C. therm polymerase from Roche, are also widely available on a commercial basis.
Assorted other agents or compounds are sometime added to the reaction to achieve the desired results. For example, DMSO can be added to the reaction, but is reported to inhibit the activity of Taq DNA Polymerase. However, DMSO has been recommended for the amplification of multiple template sequences in the same reaction. See Innis et al. Stabilizing agents such as gelatin, bovine serum albumin, and non-ionic detergents (e.g. Tween-20) are commonly added to amplification reactions. See Innis et al. For the amplification of class II sequences, the addition of 0.2% Triton X-100 has been found to be preferred. In addition, to enhance specificity by decreasing spurious priming, methods that incorporate xe2x80x9chot startxe2x80x9d (e.g., AmpliWax(copyright) (Applied Biosystems, Inc.), or an monoclonal antibody to Taq polymerase (CLONTECH Laboratories, Inc.) can be used to increase the specificity of an amplification reaction.
Amplification Programs
To amplify the HLA gene sequences of interest, the amplification reaction mixture is subjected to a series of temperatures to repeatedly denature the nucleic acid, anneal the oligonucleotide primers, and extend the primers with the polymerase. The use of a thermal cycling device can greatly facilitate the temperature cycling required in certain embodiments of the present invention. The optimum denaturing, annealing and extending temperatures can be determined by one of skill in the art for a particular oligonucleotide primer pair(s) and HLA gene template(s). In general, the extension step is carried out at a temperature of about 72xc2x0 C. and the denaturing step is carried out at about 96xc2x0 C. In addition, it may be necessary to carry out different sets of amplification cycles in succession to achieve the desired results. In addition, the number of cycles is an important consideration. Typically, one of skill in the art can carry out experiments to determine what is the optimum number of cycles to amplify the desired template(s).
The annealing temperature is of critical importance in any amplification reaction. If the annealing temperature is too low, non-specific amplification of undesired templates can arise. If the annealing temperature is too high, the template may not be efficiently amplified if at all. Determining the optimum annealing temperature for in reactions that involve large numbers of different oligonucleotide sequences and HLA templates is particularly important. A preferred amplification program for amplifying template HLA gene sequences where both primers are in solution is the following 6-stage program:
A number of controls can be used in the amplification methods described herein. They include, but are not limited to: 1. Omission of Primersxe2x80x94Control of spurious priming; 2. Known negative controlxe2x80x94Control of specificity; 3. Known positive controlxe2x80x94Control of sensitivity; 4. Omission of DNA Polymerasexe2x80x94Detection of nonspecific probe and/or enzyme/antibody sticking; 5. Use of irrelevant probes for hybridizationxe2x80x94Control for hybridization; 6. Amplification of endogenous control DNA sequencexe2x80x94Detection of false negatives, control of DNA/RNA quality.
V. Washing
After a hybridization step or after solid-phase PCR (e.g., amplification with an immobilized primer), a solid phase can be washed with a buffer to decrease non-specific binding, to wash away unbound primers, or to provide a solution that is more appropriate for subsequent detection of a detectable label, etc. Where an oligonucleotide has been immobilized or hybridized to an oligonucleotide on a solid support, the unbound oligonucleotides can be washed from a bound complex using variety of separation methods known in the art. There are many separation methods known in the art (e.g., filtering, sedimenting, centrifuging, decanting, precipitation, etc.) that can be used or adapted for use in the present invention. For example, where the amplification product is immobilized on a microtiter plate, the unbound oligonucleotides can be aspirated from the well, leaving behind those amplification products, HLA allele sequences, etc. that are bound to a solid phase. Another separation method is the immobilization of an amplification product, HLA allele sequence, etc. on a paramagnetic bead, and the decantation or aspiration of the unbound primers and oligonucleotides leaving behind the bound complex containing a detectable label remaining on the solid phase. Commercial kits, methods and systems are commercially available and can be adapted or used with the present invention (e.g., the KingFisher(trademark) system from Thermo Labsystems, Inc.).
A wash buffer can contain a detergent, or other agents, and compositions that are compatible with retention of the bound complex on the solid phase. A blocking agent is generally present in the wash buffer. Blocking agents include, but are not limited to non-fat dry milk, herring sperm DNA, dextran sulfate, and BSA. For example, a wash buffer that can be used in the present invention is a solution of 0.1% BSA in PBS. The use of 0.1% BSA results in optimum results. One or more washes may be necessary to achieve optimum lowering of non-specific binding.
VI. Detection
A wide range of methods can be used to detect the presence of oligonucleotides that contain a detectable label. The method of detection depends on the nature of the detectable label that is present. If the label is directly or indirectly capable of generating a signal in the visible light range, then a spectrophotometer can be used. Similarly, a fluorescent detectable label or signal generated therefrom can be measured using a fluorescent spectrophotometer. Alternatively, luminometers can be used to measure chemiluminescent signals. Isotopic labels can be measured using a liquid scintillation counter or in some casesxe2x80x94x-ray film. In certain embodiments, it is preferred to use a spectrophotometric plate reader that can read microtiter plates in an automated system.
VII. Analysis of Results of Assays
Computer programs containing algorithm(s) can be used to score, interpret and assign HLA alleles in certain embodiments of the present invention. Briefly, the data from a detection instrument (e.g., a spectrophotometer, an ELISA reader, a scintillation counter, etc.) can be analyzed through the use of a computer program that compares the values of each sample against a reference value(s).
For example, computer programs for the ELISA format readers take readings below a designated threshold and label such as negative and values above the same thresholds as positives. A positive well or a combination of certain wells would then represent a specific gene sequence or allele and be scored as such with the automated program. The optical density (O.D.) values obtained from reading of the wells of the ELISA plate readers are given as numerical values ranging from 0.000-2.000. This information is automatically downloaded onto the attached computers via the vendor provided software. The O.D. values are saved in a spreadsheet format in the vendor provided program as raw data.
The first step in computer analysis of the data is to validate and assign the negative control reading from the negative control well, which always exists in the same well location on the plate. A properly performed negative control is assigned as the negative value. In some embodiments where peroxidase is used with TMB, negative controls are deemed properly performed when the O.D. values are below 0.2. The usual O.D. values of a negative control reaction yielding colorless products are usually between 0.05 and 0.1. Then the threshold level is determined for that particular reaction to be 3.5 times the value of the negative control. A well is considered weakly positive if the reaction yields an O.D. reading that exceeds 3.0-fold but is below 3.5-fold of the negative control reading. A weakly positive well is rejected if two other strongly positive alleles are present for that locus. In the absence of two other strongly positive alleles for each locus, the weakly positive well is accepted if it is confirmed with repeat testing or alternative methods. A truly positive well is assigned when the O.D. readings exceed 3.5-fold over the value of the negative control. The computer program analyzes the results of all the wells, determines the positive wells based on the established criteria, and assigns the alleles based on which primer pairs exist in the positive wells. If more than two alleles are identified per locus, then the results have to be analyzed using the following protocol and confirmed by repeat testing or alternative methods.
By storing numerical reading values for the various primer pairs, many different type of assessment are possible. For example, the effects of the changes in primer pairs and primer sequences on average O.D. readings can be assessed. Consistently weak reacting sets can be replaced with primer pairs giving more robust and consistent results. Alternatively, if a particular weak reacting set of primers have no substitute, then handicap scores can be given. A more consistent tray can be developed by using the reading values as a point of reference.
VIII. High Throughput Methods and Systems
In the present invention, high-throughput analysis of HLA genotypes can be performed using automated devices. For example, an automated workstation (see e.g., U.S. Pat. No. 5,139,744, xe2x80x9cAutomated laboratory workstation having module identification meansxe2x80x9d) can be used to perform many of the steps involved in the present invention. An xe2x80x9cautomated workstationxe2x80x9d is typically a computer-controlled apparatus which can, through robotic functions, transfer, mix, and remove liquids from microtiter plates. An automated workstation can also contain a built-in plate reader, which can read the absorbance of a liquid in a microtiter well. The automated workstation can be programmed to carry out a series of mixing, transfer, and/or removal steps. The automated workstation will typically have a multi-channel pipettor which can pipette small amounts of liquid (e.g., microliter amounts) from a vessel to the well.
For example, in some embodiments of the present invention, the automated workstation can be used to transfer DNA samples, oligonucleotides, amplification reagents. The automated work station can also be used to wash the samples using wash buffer. In addition, detection of oligonucleotides containing a detectable label can be carried out using an automated workstation. For example, the automated workstation can be used to add a detection reagent to the wells. The automated workstation, when equipped with a plate reader, can monitor the absorbance of the reaction of the detection reagent in the wells.
A number of robotic fluid transfer systems/automated work stations are available, or can easily be made from existing components. For example, a Zymate XP (Zymark Corporation; Hopkinton, Mass.) automated robot using a Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous ligation reactions. Other automatic microplate dispensers include Lambda Jet and Lambda Dot (One Lambda, Inc. CA), and various other automatic plate washers and dispensers (e.g. from Thermo Labsystems, Inc. or Molecular Devices, Inc.). Moreover, it will be apparent to those of skill in the art that the PCR setup, reagent addition and washing steps can be automated with existing robotics outlined above.
Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intel x86 or Pentium chipxe2x80x94compatible DOS OS2 WINDOWS, WINDOWS NT or WINDOWS 98 based machines), MACINTOSH, or UNIX based (e.g., SUN work station) computers.
One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques. The use of such automated machines, can minimize the existence of false positives, labor requirements, variabilities, human errors, human subjectivity, and human expertise requirements, and maximizes throughput, accuracy, sensitivity and specificity.
IX. Hybridization of Capture Oligonucleotides to HLA Amplification Products
Hybridization of Immobilized Capture Oligonucleotides to HLA Amplification Products
This method involves the use of immobilized oligonucleotides to capture HLA allele sequences contained in an amplification product. Briefly, HLA allele sequences are amplified from a template nucleic acid using HLA allele-specific forward and reverse primers. One or both of the amplification primers can contain a detectable label. Then the amplification products are denatured and hybridized to a locus-specific or allele-specific capture oligonucleotide that is already immobilized to a solid phase to form a detectable complex. The presence of the detectable label in the detectable complex is then measured using methods known to those of skill in the art (e.g., spectrophotometric means, a luminometer, etc.), which may require the addition of one or more detection reagents (e.g., an avidin-enzyme molecule with a colorimetric enzyme substrate).
The capture oligonucleotides possess sufficient nucleotide complementarity to the HLA allele sequences being amplified that they can hybridize to them under stringent conditions. Typically, the HLA allele-specific forward and/or reverse primer will contain a detectable label (e.g., biotin, digoxigenin, EDANS, or a fluorescent moiety, etc.) so as to facilitate detection. Thus, this method allows for the amplification of many different HLA alleles which can be detected with, in the case of some HLA loci, as little as one capture oligonucleotide that is locus-specific. This is an advantage over previous methods, in which allele-specific capture oligonucleotides were used, as the detection of hundreds of alleles would require hundreds of allele-specific capture oligonucleotides (see e.g., Erlich et al. (1991) Eur. J. Immunogenet. 18(1-2): 33-55; Kawasaki et al. (1993) Methods Enzymol. 218:369-381). Thus, the present invention permits a great simplification and reduction in the number of oligonucleotides required to detect hundreds of HLA-alleles.
Hybridization of Free Capture Oligonucleotides to HLA Amplification Products and Subsequent Immobilization of the Detectable Complex
In another embodiment of the present invention, the hybridization takes place in solution with capture oligonucleotide(s) and then the capture oligonucleotide is immobilized. This method involves the use of capture oligonucleotides that are hybridized in solution to HLA allele sequences contained in an amplification product and subsequent immobilization of the capture oligonucleotide to a solid phase. First, HLA allele sequences are amplified from a nucleic acid using HLA allele-specific forward and reverse primers. Then the amplification product are denatured and hybridized to a locus-specific or allele-specific capture oligonucleotide that is already immobilized to a solid phase. The capture oligonucleotides then hybridize and bind to the denatured single stranded PCR products at a suitable hybridization temperature and xe2x80x9ccapturexe2x80x9d complementary sequences in the products onto the plate. If none or very little complementary sequences for the capture oligonucleotide are present after the nucleic acid amplification reaction (for example, if the allele sequence represented by the allele-specific PCR primers are not present in the sample DNA template, then no PCR product would be formed), then it is unlikely a detectable complex will form. The capture oligonucleotides possess sufficient nucleotide complementarity to the HLA allele sequences being amplified that they can hybridize to them under stringent conditions. Typically, the HLA allele-specific forward and/or reverse primer will contain a detectable label (e.g., biotin, digoxigenin, EDANS, or a fluorescent moiety, etc.) so as to facilitate detection.
In this method, capture oligonucleotides with either conserved sequences (e.g., locus-specific oligonucleotides) or allele specific sequences can be used. The later offering an additional level of specificity whereas the former offers convenience and ease of setup as well as lower cost in having fewer sets of oligonucleotides. Thus, this method allows for the amplification of many different HLA alleles which can be detected with, in the case of some HLA loci, as little as one capture oligonucleotide that is locus-specific. This is an advantage over previous methods, in which allele-specific capture oligonucleotides were hybridized in solution to a locus-specific HLA amplification product, as the detection of hundreds of alleles would require hundreds of allele-specific capture oligonucleotides (see e.g., Nevinny-Stickel and Albert (1993) Eur. J. of Immunogenet., 20: 419-427). Thus, the present invention permits a great simplification and reduction in the number of oligonucleotides required to detect hundreds of HLA-alleles.
X. Amplification of HLA Sequences with Immobilized Primers
This method involves the amplification of HLA sequences using allele-specific primers, where one of the pair of amplification primers is immobilized to a solid phase. The other primer constituting the primer pair contains a detectable label and is initially free in solution. This technique is not limited to the detection of HLA alleles. Essentially, any set of amplification primers and any gene can be amplified. With this method, the immobilized amplification primer serves to immobilize the amplification product directly to a solid phase. The amplification should only take place if allele that can be amplified with a particular pair of allele-specific primers is present in solution. The nucleic acid amplification and capture of PCR product take place on the same polycarbonate plate and the capture oligonucleotide/PCR primer is an allele specific sequence that identifies the sequence of interest (e.g. the particular HLA allele) and serves three purposes. First, it serves as the capture oligonucleotide and immobilizes the PCR products onto the plates. Second, it serves as one of the PCR primers that facilitate the nucleic acid amplification reaction. Third, it serves as the discriminating sequence that allows identification of the correct allele. This means that the PCR amplification reaction would only take place if the correct sequences that is perfectly complementary to the template (which is the particular allele of the person whose HLA sequence or other sequence is being typed) is present on both PCR primers. An advantage of this method is the elimination of transfer, reduction of an additional set of oligonucleotides to the assay vessel (compared with two previous methods described under Section X).
If a sequence specific nucleic acid amplification reaction occurred due to perfect matching between the PCR primers and the template sequences, then the product would be immobilized on the solid phase. Following capture, the unbound non-specific labeled PCR primers can be washed off. With fluorescent probes, the plate can be read with an automated fluorescent ELISA format reader. With calorimetric reactions that are associated for example with avidin conjugated enzyme and substrate systems (e.g. avidin-conjugated horseradish peroxidase and TMB), a photometric ELISA format reader would be able to quantitate the result.
XI. Multiplexing of Positive Controls
In certain embodiments, one or more positive control can be added to each reaction vessel. For example, a positive control in every well can be used to distinguish from the allele specific reactions by virtue of having a different fluorophore or enzyme-substrate combinations. For example, if the allele specific reaction and the positive control use different fluorophores, then the excitation and emission wavelengths for both fluorophores can be used. The positive control amplified fragment would be longer than the allele specific reaction so that the allele specific reaction would be favored. The positive controls would be captured by the same capture probe as the allele specific if the capture probe is locus-specific. If allele-specific capture probes are used, then the positive controls may have complementary sequences to the allele specific capture probes at its 5xe2x80x2 end of the primer that is labeled.
XII. Magnetic Bead Variation
This method takes advantage of a commercially available nucleic acid purification method that employs magnetic beads coated with avidin or other materials to facilitate the xe2x80x9cfishingxe2x80x9d of the appropriate nucleic acid product of interest (KINGFISHER(trademark) available from Thermo Labsystems, Inc.). For example, if biotinylated oligonucleotide PCR primers are used, then a biotinylated PCR product will be captured with the avidin on the beads. The magnetic beads are then pulled out of the reaction well, washed and all non-biotinylated materials will be washed off. The biotinylated products and primers are then separated from avidin coated beads by further treatment, such as elution with excess free biotin. Thereafter, the biotinylated products are hybridized to the capture probe of interest and separated from the biotinylated primers. Alternatively, a labeled hybridization probe is allowed to bind to the PCR product, followed by washing using the KINGFISHERTM method to remove any unbound non-specific signals. Lastly, the signals would be measured. Instead of biotinylated beads, covalently modified beads that attach to PCR oligonucleotides can also be used.
XIII. SSOP with Molecular Beacon Detection
In the methods of the present invention, molecular beacon oligonucleotides can be used to hybridize with allele-specific amplification products. Once the modular beacons are hybridized to a complementary sequence in an amplified product, the quencher group is no longer close enough to quench the fluorophore. As a consequence the fluorophore can be detected and quantitated. These molecular beacon oligonucleotides are known in the art and can be readily designed (see Materials section on design and construction of molecular beacon oligonucleotides). These oligonucleotides have the advantage of being directly assayable with a device that can measure fluorescence. In addition, this method can exhibit lower background signal than other methods as only oligonucleotides that are incorporated into an allele-specific product will give off a signal. Thus, molecular beacon detection does not require the addition of a detection reagent to observe whether an HLA genotype is present in an analyte.
XIV. In Situ Amplification Variation
In certain embodiments, the in situ amplification method is chosen to eliminate the need for DNA extraction and preparation. In contrast to the usual limitations of in situ amplification where the number of cycles has to be curtailed to prevent the floating away of amplified products from the cell, it is irrelevant whether amplified product stays in the cell or out. As a result, the same number of cycles can be used to generate the same degree of amplification as traditional PCR. If molecular beacon method is not incorporated into the protocol, then the reaction products from the wells will be transferred to another microtiter plate that has surface attached capture oligonucleotide probes that are similar to the ones described earlier with either conserved sequences which can be used in all the wells or allele specific sequences. By using an in situ amplification method it is then possible to use molecular beacons to detect the amplified products. In situ amplification can be carried out on a microscopic slide, a tissue sample, a microtiter plate, etc.
The molecular beacon method can be incorporated to eliminate even the washing step as well as the need for specially modified plates that can be quite expensive. It also allows for real time measurement of PCR product formation. When PCR products are formed and denatured during the various cycling steps, molecular beacons would hybridize to some of the complementary single strands, thereby fluorescing and allowing real time measurement. If real time measurement is not desired, then the molecular beacon probe can be added at the end of the reaction and only wells with amplified products that are complementary to the molecular beacon would light up. Because the unbound molecular beacon does not fluoresce, washing steps may not be necessary if the signal to noise ratio is high enough.
XV. Tissue Block Section Variation
The methods of the present invention can be carried out on paraffin embedded formaldehyde fixed sections of buffy coats, umbilical cord blood clots or blood clots placed onto glass slides with grids. The same sample can be placed onto one slide and different probes are used in an in situ method or many samples can be placed onto the same slide and the same probe is used for all the samples. In the latter, as many sections and slides of the samples will be cut as the number of probes plus controls. This method appears to be easier for the amplification, since there is no need to separate the different probes or reactions from one another.